Echography of the Cervix and Uterus During the Proliferative and Secretory Phases of the Menstrual Cycle in Bonnet Monkeys (Macaca Radiata)

Home , Cervix

Echography of the Cervix and Uterus during the Proliferative and Secretory Phases of the Menstrual Cycle in Bonnet Monkeys (Macaca radiata)

Uddhav K Chaudhari,1,* Siddnath M Metkari,2 Dhyananjay D Manjaramkar,2 Geetanjali Sachdeva,1 Rajendra Katkam,1 Atmaram H Bandivdekar,3 Abhishek Mahajan,4 Meenakshi H Thakur,4 and Sanjiv D Kholkute1

We undertook the present study to investigate the echographic characteristics of the uterus and cervix of female bonnet monkeys (Macaca radiata) during the proliferative and secretory phases of the menstrual cycle. The cervix was tortuous in shape and measured 2.74 ± 0.30 cm (mean ± SD) in width by 3.10 ± 0.32 cm in length. The cervical lumen contained 2 or 3 colliculi, which projected from the cervical canal. The echogenicity of cervix varied during proliferative and secretory phases. The uterus was pyriform in shape (2.46 ± 0.28 cm × 1.45 ± 0.19 cm) and consisted of serosa, myometrium, and endometrium. The endometrium generated a triple-line pattern; the outer and central lines were hyperechogenic, whereas the inner line was hypoechogenic. The endometrium was significantly thicker during the secretory phase (0.69 ± 0.12 cm) than during the proliferative phase (0.43 ± 0.15 cm). Knowledge of the echogenic changes in the female reproductive organs of bonnet monkeys during a regular menstrual cycle may facilitate understanding of other physiologic and pathophysiologic changes.

Ultrasound imaging is a noninvasive, atraumatic, and simple Materials and Methods method to assess various organs in humans and nonhuman pri- Animals and husbandry practices. Adult female bonnet macaques 11 mates. Ultrasonography has been used to detect fetal growth, (Macaca radiate; n = 15) showing normal menstrual cycles under- endometrial thickness, and endometriosis in rhesus macaques went abdominal ultrasonography during their late proliferative 17,20,26 (Macaca mulatta), and color Doppler ultrasonography (days 6 through 13) and midsecretory (days 16 through 21) phases. has used for characterizing uteroplacental hemodynamics and Bonnet monkeys used in the present study were wild-caught from 22,29 assessing fetal and placental blood flow in that species. In a forest in southern India. Monkeys were housed individually and cynomolgus macaques (Macaca fascicularis), ultrasound tech- fed with fresh seasonal vegetables, fruits, ground nuts, bangal niques have been applied in transfundal uterine sperm recovery, grams, and so forth. Water was provided ad libitum. Housing follicular fluid aspiration, assessment of endometrial changes conditions were a 12:12-h light:dark cycle, with a temperature of during the menstrual cycle, evaluation of folliculogenesis, 24 to 28 °C and relative humidity of 30% to 70%. Previous reports 5,33-35 and ovulation induction studies. Other primates species from our laboratory have shown that the length of menstrual wherein ultrasound techniques have been used are vervet mon- cycle in bonnet monkeys is 25 to 30 d.6,24 Menstrual cyclicity was 31 30 keys (Cercopithecus aethiops), owl monkeys (Aotus nancymaae), monitored for at least 2 cycles by daily vaginal swab examination 9,12 and common marmosets (Callithrix jacchus). at 1100. The day of visible vaginal bleeding was considered as Bonnet macaques (Macaca radiata) have been used as an animal day 1, and ultrasonography was performed on days 6 through 6,23,24 model for research on contraceptive agents, endometrial 13 (proliferative phase) and 16 through 21 (secretory phases). The 18,27,28 10 biology, and assisted reproductive technologies. Al- ultrasound protocol was approved by the Institutional Animal though bonnet macaques bear close similarity with rhesus Ethics Committee, and ultrasound examinations were performed macaques, ultrasound profile of cervix and uterus in these spe- during routine health monitoring of bonnet monkeys in the colony. cies has not yet been characterized. We undertook the present To ensure that the monkey’s urinary bladders should be full dur- study to evaluate the echographic profile of the cervix and ing ultrasound examination, water bowls were removed during uterus in regularly cycling bonnet macaques in the proliferative the evening prior to the exam and replaced the morning of the (days 6 through 13 after overt bleeding) and secretory phases ultrasonography. The animal facility at the institute is registered (days 16 through 21) of menstrual cycle. Knowledge of the nor- with the Committee for the Purpose of Control and Supervision of mal ultrasonographic profiles of the female reproductive organs Experiments on Animals, Ministry of Social Justice and Empower- may facilitate the identification of pathophysiological changes ment, Government of India (registration no. 78/1999/CPCSEA). during natural or stimulated cycles in bonnet monkeys. Gynecologic ultrasound examination. A gynecologic ultrasound evaluation of the cervix and uterus was performed in B mode by Received: 22 May 2013. Revision requested: 03 Jul 2013. Accepted: 11 Jul 2013. using high-resolution linear curved transducers (L12-5 MHz and 1Department of Primate Biology, 2Animal House Facility, and 3Department of Biochem- C5-2 MHz probes, HDXE11 ultrasound system, Philips, Andover, istry, National Institute for Research in Reproductive Health (NIRRH), Indian Council MA). The shape, echogenic texture, position, and dimensions of of Medical Research (ICMR), Parel, Mumbai, India; 4Department of Radiodiagnosis, Tata Memorial Hospital (TMH), Parel, Mumbai, India. the cervix and uterus were studied during the late proliferative *Corresponding author. Email: [email protected] and midsecretory phases of the menstrual cycle. Monkeys were

18 Ultrasonography of reproductive organs

Figure 1. An ultrasound image of the reproductive organs of bonnet monkeys obtained by using the C5-2 probe during (A) late proliferative and (B) midsecretory phases of the menstrual cycle, demonstrating the echogenicity of the uterus and cervix during the proliferative (hypoechoic) and midsecretory (hyperechoic) phases. The cervical canal appears open and filled with mucus during the proliferative phase compared with secretory phase. C, cervix; CC, cervical canal; CrDC, cranial dorsal colliculus; CuDC, caudal dorsal colliculus; Is, isthmus; U, uterus; UC, uterine cavity; V, vagina; VC, ventral colliculus.

Figure 2. An ultrasound image of the cervix of bonnet monkeys during the late proliferative phase of the menstrual cycle, obtained by using the L12-3 probe. (A) Sagittal view. (B) Transverse view. The cervical canal appears open and contains mucus, and colliculi are separated and distinctly visible. CC, cervical canal; CrDC, cranial dorsal colliculus; CW, cervical wall; VC, ventral colliculus. sedated with ketamine (10 mg/kg body weight) and placed in a RWave Cardio Products, Mumbai, India) was applied to the shaved supine position. The region was disinfected (Savlon, Johnson and area. Initially the entire reproductive tract was examined by using Johnson, Mumbai, India), and the hair was shaved from 2/3 of the the C5-2MHz transducer. The transducer was placed sagittally abdominal cavity, to the pubic symphysis. Ultrasound gel (RTrace, on the lower midline of the pelvic–abdominal region to locate the

19 Vol 53, No 1 Journal of the American Association for Laboratory Animal Science January 2014

Figure 3. An ultrasound image of the cervix of bonnet monkeys during the midsecretory phase of the menstrual cycle, obtained by using the L12-3 probe. (A) Sagittal view. (B) Transverse view. The cervical canal appears collapsed, and colliculi are merged. CC, cervical canal; CrDC, cranial dorsal colliculus; CuDC, caudal dorsal colliculus; VC, ventral colliculus. vagina, cervix, and uterus. The linear L12-5 MHz transducer then Table 1. Cervical, uterine, and endometrial measurements (cm; mean was used for individual examination of the cervix and uterus. The ± SD) during the late proliferative and midsecretory phases of the uterus was localized in the lower 2/3 of the abdominal area. The menstrual cycle of bonnet monkeys cervix was localized above the pubic symphysis. The craniocaudal Late proliferative Midsecretory and dorsoventral diameters of the cervix, uterus, and endometrium Cervix were measured from sagittal scans, and the transverse diameters of Craniocaudal diameter 2.74 ± 0.30 2.69 ± 0.24 these organs were obtained from transverse scans.16 Four observa- Dorsoventral diameter a b tions were made for each monkey during both the proliferative and 3.10 ± 0.32 2.88 ± 0.37 secretory phases of the menstrual cycle. The cervix was measured Transverse diameter 2.86 ± 0.28 2.62 ± 0.27 from the internal os to external os (craniocaudal diameter) and from Uterus the dorsal colliculi to ventral colliculi (dorsoventral diameter). The Craniocaudal diameter 2.46 ± 0.28 2.41 ± 0.32 uterus was measured from the outer body of uterus (fundus) to the Dorsoventral diameter 1.45 ± 0.19a 1.39 ± 0.24b internal os of the cervix to obtain the craniocaudal and dorsoventral Transverse diameter 1.42 ± 0.37 1.52 ± 0.42 diameters (extending from the dorsal to the ventral side of uterus). Endometrium The endometrium appeared as a triple-line pattern consisting of Craniocaudal diameter 1.20 ± 0.23 1.34 ± 0.42 a prominent outer and central hyperechogenic lines and an inner Dorsoventral diameter 0.43 0.15c 0.69 0.12c hypoechogenic or black region on sagittal and transverse views. ± ± d d The central hypoechogenic region of the endometrium was meas- Transverse diameter 0.56 ± 0.85 0.79 ± 0.14 ured on sagittal scans as the craniocaudal (longitudinal) diameter. Data were obtained by using the L12-3 probe during sagittal and Endometrial thickness (dorsoventral diameter) was determined by transverse scans. measuring the distance between the outermost interfaces of both Values indicated by the same lowercase letter differ significantly (P < sides of the echogenic interface adjacent to myometrium. The color 0.05, Student t test). Doppler mode was used to assess the vascularity of the uterus during the proliferative and secretory phases of menstrual cycle. MHz probe can scan the entire reproductive system for localiza- Statistical analysis. Measurements of cervix and uterus ob- tion of the vagina, cervix, and uterus. The L12-3 MHz transducer tained during ultrasonography are expressed as mean ± 1 SD. is a high-resolution linear probe that allows excellent evaluation Endometrial thickness during the proliferative and secretory of the echogenic texture of the cervix, uterus, and endometrium. phases was compared by using the Student t test (Prism 5, Cervix. Both probes were used to assess the entire cervical GraphPad Software, San Diego, CA). canal ultrasonographically (Figures 1 through 3). Table 1 sum- marizes the cervical measurements. The cervix was broader (P Results < 0.05) than the uterus. The cervical canal contained several In the present study, the cervix and uterus of 15 bonnet mon- turns, giving rise to a tortuous shape. The 3 collicular struc- keys (average weight, 5.1 ± 1.3 kg) with regular menstrual cycles tures were attached by broad bases to the cervical walls. A large were evaluated by ultrasonography during the late prolifera- prominent ventral colliculus and the intermediately sized tive (days 6 through 13) and midsecretory (days 16 through 21) cranial dorsal colliculus were readily identified as discrete phases. We used 2 different transducers for this purpose. The C5-2 structures near the ectocervix. The third, caudal dorsal col-

20 Ultrasonography of reproductive organs

Figure 4. An ultrasound image of the uterus and endometrium of bonnet monkeys during the late proliferative phase of the menstrual cycle, obtained by using the L12-3 probe. (A) Sagittal view. (B) Transverse view. The uterus appears in a typical triple-line pattern consisting of a prominent outer and centrally hyperechogenic line (endometrium) and an inner, hypoechogenic or black region (uterine cavity) separated by homogeneously grayish myometrium. E, endometrium; M, myometrium; U, uterus; UB, urinary bladder; UC, uterine cavity.

Figure 5. A sagittal view of a color Doppler ultrasound image of the uterus of bonnet monkeys during the (A) late proliferative and (B) midsecretory phases of the menstrual cycle, obtained by using the L12-3 probe. The uterus demonstrated increased vasculature and blood flow during the proliferative phase compared with the secretory phase. E, endometrium; IA, iliac artery; M, myometrium; U, uterus; UA, uterine artery. liculus merged with the fibrous dorsal lip of the external os of Uterus. The uterus was located centrally in the abdominal the cervix (Figure 1 A and B). The ventral and cranial dorsal cavity, below the urinary bladder. On sagittal scans, the uterus collicululi were demarcated easily on sagittal and transverse measured 2.5 ± 0.28 cm × 1.45 ± 0.20 cm (length × width) and scans during the proliferative phase (Figure 2 A and B). Dur- appeared pyriform in shape (Figure 4 A); on transverse scans, ing the secretory phase of the menstrual cycle, the ventral and the uterus was oval in shape (Figure 4 B). The myometrium was cranial dorsal colliculi were hyperechoic and appeared merged homogeneously echodense. The inner layer of myometrium on sagittal and transverse scans (Figure 3 A and B). In addition, was moderately echogenic, but the junctional zones of the en- the echogenicity and texture of the cervix changed during the dometrium were well demarcated and smooth in outline (Figure proliferative and secretory phases of the menstrual cycle. The 4 A and B). Color Doppler ultrasonography revealed the location walls and cervical canal were distended and hypoechoic dur- of the uterine artery on the left, at the junction of the cervix and ing the proliferative phase (Figure 2 A and B); this appearance uterine corpus (Figure 5 A). Blood flow and vascularity in the likely was due to excessive cervical secretions. The cervix was uterus were increased during the proliferative phase (Figure 5 hyperechoic and the cervical canal was collapsed during the A) compared with the secretory phase (Figure 5 B). secretory phase (Figure 3 A and B). Endometrium. On sagittal and longitudinal views, the uterine endometrium had a triple-line pattern, with 2 prominent, outer

21 Vol 53, No 1 Journal of the American Association for Laboratory Animal Science January 2014

Figure 6. An ultrasound image of the uterus and endometrium of bonnet monkeys during the midsecretory phase of the menstrual cycle, obtained by using the L12-3 probe. (A) Sagittal view. (B) Transverse view. The uterus appears as homogeneously echogenic, with absence of the characteristic triple-line pattern. E, endometrium; M, myometrium; UC, uterine cavity. and central hyperechogenic lines and an inner hypoechogenic or and mucus secretion of the cervix change during the proliferative black region (Figures 4 and 6). During the proliferative phase, the and secretory phases of menstrual cycle in macaques and humans. endometrium appeared more echogenic than was the myometrium The quantity of cervical mucus secretion increases proportionately and developed a multilayered appearance, in which an echogenic with plasma estrogen concentration in bonnet monkeys14 and basal layer and hypoechoic inner functional layer were separated humans.13 We noted that the cervical walls of bonnet monkeys by a thin echogenic median layer of uterine cavity (Figure 4 A and are hypoechoic during the proliferative phase, suggesting actively B). The echogenicity of the endometrium increased during the secretion into the lumen of the cervix. In addition, we found that secretory phase as the multilayer pattern disappeared (Figure 6 the cervical canal was open and completely echoic, indicating A and B). Endometrium thickness was measured from echogenic hydrated mucus accumulation. Compared with rhesus macaques, border to echogenic border, across the endometrial cavity, on a bonnet monkeys secrete very high amounts of cervical mucus.21 sagittal midline image. The endometrium was significantly P( < Under the influence of estrogen, the cervical canal widens due to 0.005) thicker during the secretory phase (6.9 ± 0.8mm) as compared accumulation of cervical secretions in dogs,32 cats,1 and humans.4 with during the proliferative phase (4.3 ± 1.1 mm; Table 1). The endometrium of bonnet monkeys proliferates during the proliferative phase (days 6 through 13) and differentiates dur- Discussion ing the secretory phase (days 16 through 21) of the menstrual South Indian bonnet monkeys are closely similar to rhesus cycle. During the proliferative phase and as a result of estrogen macaques. Previous reports from our laboratory showed that the signaling, the endometrium develops glands, blood vessels, and length of the menstrual cycle in bonnet monkeys is 25 to 30 d.6,24 stroma.31 Ultrasound examination of the endometrium during Menstrual cyclicity reflects cyclic changes in the levels of reproduc- the proliferative phase was found it to be echogenic relative to tive hormones, such as estrogen and progesterone. Endometrial the myometrium. A typical multilayer endometrium consists of proliferation, ovarian follicular development, cervical mucus se- an echogenic basal layer and hypoechoic inner functional layer, cretions are associated with the menstrual cycle phase in bonnet which are separated by a thin echogenic median layer arising monkeys.14 The gross anatomy of cervix uteri has been compared from the luminal content. In rhesus macaques, the reflectivity among nonhuman primates. The cervical canal of bonnet and (echogenicity) of the endometrium varies according to the stage rhesus macaques have several sharp turns due to the protrusion of of the menstrual cycle; the maximal thickness of the endometrium colliculi, giving rise to the characteristic tortuous shape to the cer- was 8 mm during the secretory phase.17 We similarly observed vix.8 Vaginal hysterography of bonnet monkeys and contrast films highly echogenic endometrium during the proliferative phase of excised uteri have shown the similar cervical morphology in the of the menstrual cycle and maximal endometrial thickness (0.69 bonnet and rhesus macaques.23 Our ultrasound observations are in ± 0.12 cm) during the secretory phase. Color Doppler imaging agreement with previous investigations in bonnet monkeys. The showed increased vascularity and blood flow during the prolif- exact role of these colliculi is not known. However, microscopically, erative phase compared with the secretory phase. Endometrial colliculi consist of columnar epithelium, and they support mucus echogenicity and thickness are associated with pregnancy out- secretion during the estrogen-dominant proliferative phase of the comes in humans.2,25 A nonhomogeneous hyperechoic (rather menstrual cycle.21 In addition, the cervical colliculi may act as a than a triple line) pattern of the endometrium before pregnancy sperm reservoir and, alternatively, as a barrier to prevent excessive is associated with lower pregnancy rates.2 Several studies in spermatozoa from reaching the site of fertilization. Motile sperm humans have suggested a correlation between endometrial thick- have been recovered from the cervix of humans,7 cattle,15 and ness and endometrial receptivity during autologous,36 oocyte marmosets,8 2 to 5 d after insemination. The echogenicity, texture, donor,3 and in vitro fertilization cycles.19

22 Ultrasonography of reproductive organs

The present study reports the echogenic changes of the 18. Nimbkar-Joshi S, Katkam RR, Chaudhari UK, Jacob S, Manjram- cervix and uterus of bonnet monkeys during the proliferative kar DD, Metkari SM, Hinduja I, Mangoli V, Desai S, Kholkute and secretory phases of the menstrual cycle. This information SD, Puri CP, Sachdeva G. 2012. Endometrial epithelial cell may help in the delineation of pathologic conditions in these modifications in response to embryonic signals in bonnet monkeys (Macaca radiata). Histochem Cell Biol 138:289–304. organs and the selective enrolment of macaques for cervical 19. Noyes N, Hampton BS, Berkeley A, Licciardi F, Grifo J, Krey L. and endometrial research. In addition, our findings likely will 2001. Factors useful in predicting the success of oocyte donation: facilitate the evaluation of female reproductive organs during a 3-year retrospective analysis. Fertil Steril 76:92–97. gonadotropin stimulation and the development of appropriate 20. Nyland TG, Hill DE, Hendrickx AG, Farver TB, McGahan JP, nonhuman primate models for reproductive disorders. Henrickson R, Anderson J, Phillips HE. 1984. Ultrasonic assessment of fetal growth in the nonhuman primate (Macaca mulatta). J Clin Ultrasound 12:387–395. Acknowledgments 21. Ovadia J, Mcarthur JW, Kopito L, Ulfelder H. 1971. The cervical We thank ICMR for financial support and Mr ML Shinde and Mr JS mucus secretion of the bonnet monkey (M. radiata): anatomical Lokhande for their help in animal handing during ultrasound procedures. basis and physiological regulation. Biol Reprod 5:127–145. We also thank Dr Rohit Dhumal for helping with statistical analysis. 22. Panigel M, Dixon T, Constantinidis I, Sheppard S, Swenson R, McLure H, Campbell WE, Huddleston J, Polliotti B, Nahmias References A. 1993. FAST scan magnetic resonance imaging and Doppler 1. Chatdarong K, Kampa N, Axner E, Linde-Forsberg C. 2002. Inves- ultrasonography of uteroplacental hemodynamics in the rhesus tigation of cervical patency and uterine appearance in domestic cats monkey (Macaca mulatta). J Med Primatol 22:393–399. by fluoroscopy and scintigraphy. Reprod Domest Anim3 7:275–281. 23. Pongubala JM, Elger WA, Puri CP. 1987. Relative binding affinity 2. Check JH, Dietterich C, Luri D. 2000. Nonhomogeneous hyper- of antiprogestins ZK 98.299 and ZK 98.734 for progesterone recep- echogenic pattern 3 days after embryo transfer is associated with tors in the endometrium and myometrium of bonnet monkeys. J lower pregnancy rates. Hum Reprod 15:1069–1074. Recept Res 7:903–920. 3. Check JH, Nowroozi K, Choe J, Lurie D, Dietterich C. 1993. The 24. Puri CP, Katkam RR, D’Souza A, Elger WA, Patil RK. 1990. effect of endometrial thickness and echo pattern on in vitro fer- Effects of progesterone antagonist, lilopristone (ZK 98.734), on tilization outcome in donor oocyte–embryo transfer cycle. Fertil induction of menstruation, inhibition of nidation, and termination Steril 59:72–75. of pregnancy in bonnet monkeys. Biol Reprod 43:437–443. 4. Duijkers IJM, Klipping C. 2000. Ultrasonographic assessment of 25. Richter KS, Bugge KR, Bromer JG, Levy MJ. 2007. Relationship endocervix and cervical mucus in ovulatory menstrual cycles. Eur between endometrial thickness and embryo implantation, based J Obstet Gynecol Reprod Biol 93:13–17. on 1294 cycles of in vitro fertilization with transfer of 2 blastocyst- 5. Foster WG, Stals SI, McMahon A. 1992. A prospective analysis of stage embryos. Fertil Steril 87:53–59. endometrial cycle changes by ultrasound in the female cynomolgus 26. Rippy MK, Lee DR, Pearson SL, Bernal JC, Kuehl TJ. 1996. Iden- monkey. J Med Primatol 21:30–34. tification of rhesus macaques with spontaneous endometriosis. J 6. Gopalkrishnan K, Katkam RR, Sachdeva G, Kholkute SD, Med Primatol 25:346–355. Padwal V, Puri CP. 2003. Effects of an antiprogestin onapristone 27. Rosario GX, D’Souza SJ, Manjramkar DD, Parmar VA, Puri CP, on the endometrium of bonnet monkeys: morphometric and Sachdeva G. 2008. Endometrial modifications during early preg- ultrastructural studies. Biol Reprod 68:1959–1967. nancy in bonnet monkeys (Macaca radiata). Reprod Fertil Dev 20: 7. Gould JE, Overstreet JW, Hanson FW. 1984. Assessment of human 281–294. sperm function after recovery from the female reproductive tract. 28. Sachdeva G, Patil V, Katkam RR, Manjramkar DD, Kholkute Biol Reprod 31:888–894. SD, Puri CP. 2001. Expression profiles of endometrial leukemia 8. Hafez ESE, Jaszcza S. 1972. Comparative anatomy and histology inhibitory factor, transforming growth factor β 2 (TGFβ2), and of the cervix uteri in nonhuman primates. Primates 13:297–316. TGFβ2 receptor in infertile bonnet monkeys. Biol Reprod 65:1–8. 9. Hastings JM, Morris KD, Allan D, Wilson H, Millar RP, Fraser 29. Schmiedl UP, Komarniski K, Winter TC, Luna JA, Cyr DR, HM, Moran CM. 2012. Contrast-imaging ultrasound detects ab- Ruppenthal G, Schlief R. 1998. Assessment of fetal and placental normalities in the marmoset ovary. Am J Primatol 74:1088–1096. blood flow in primates using contrast enhanced ultrasonography. 10. Hewitson L. 2004. Primate models for assisted reproductive tech- J Ultrasound Med 17:75–80. nologies. Reproduction 128:293–299. 30. Schuler AM, Parks VL, Abee CR, Scammell JG. 2007. Ultrasono- 11. James AE Jr, Brayton JB, Novak G, Wight D, Shehan TK, Bush graphic monitoring of a spontaneous abortion in an owl monkey RM, Sanders RC. 1976. The use of diagnostic ultrasound in evalu- (Aotus nancymaae). J Am Assoc Lab Anim Sci 46:74–76. ation of the abdomen in primates with emphasis on the rhesus 31. Seier JV, Horst G, Kock M, Chwalisz K. 2000. The detection and monkey (Macaca mulatta). J Med Primatol 5:160–175. monitoring of early pregnancy in the vervet monkey (Cercopithecus 12. Jaquish CE, Toal RL, Tardif SD, Carson RL. 1995. Use of ultra- aethiops) with the use of ultrasound and correlation with reproduc- sound to monitor prenatal growth and development in the common tive steroid hormones. J Med Primatol 29:70–75. marmoset (Callithrix jacchus). Am J Primatol 36:259–275. 32. Silva LDM, Onclin K, Verstegen J. 1995. Cervical opening in rela- 13. Katz DF, Slade DA, Nakajima ST. 1997. Analysis of preovulatory tion to progesterone and oestradiol during heat in beagle bitches. changes in cervical mucus hydration and sperm penetrability. Adv J Reprod Fertil 104:85–90. Contracept 13:143–151. 33. van Diepen HA, Pansier J, Oude Wesselink P, van Drie A, van 14. Kholkute SD, Joseph R, Joshi UM, Munshi SR. 1981. Some char- Duin M, Mulders S. 2012. Noninvasive translational cynomolgus acteristics of the normal menstrual cycle of the bonnet monkey (M. model for studying folliculogenesis and ovulation using color radiata). Primates 22:399–403. Doppler ultrasonography. J Med Primatol 41:18–23. 15. Mitchell JR, Senger PL. 1985. Rosenberger. Distribution and reten- 34. VandeVoort CA, Tarantal AF. 1991. The macaque model for in vitro tion of spermatozoa with acrosomal and nuclear abnormalities in fertilization: superovulation techniques and ultrasound-guided the cow genital tract. J Anim Sci 61:956–967. follicular aspiration. J Med Primatol 20:110–116. 16. Monteiro FOB, Coutinho LN, Pompeu ESS, de Castro PG, Maia 35. VandeVoort CA, Tollner TL, Tarantal AF, Overstreet JW. 1989. CE, Pereira WLA, Vicente WRR. 2009. Ovarian and uterine ultra- Ultrasound-guided transfundal uterine sperm recovery from sonography in Aotus azarai infulatus. Int J Primatol 30:327–336. Macaca fascicularis. Gamete Res 24:327–331. 17. Morgan PM, Hutz RJ, Kraus EM, Bavister BD, Cormie JA. 1987. 36. Zhang X, Chen CH, Confino E, Barnes R, Milad M, Kazer RR. Ultrasonographic assessment of the endometrium in rhesus 2005. Increased endometrial thickness is associated with improved monkeys during the normal menstrual cycle. Biol Reprod 36: treatment outcome for selected patients undergoing in vitro ferti- 463–469. lization–embryo transfer. Fertil Steril 83:336–340.